Expression of the zinc-finger gene PLZF at rhombomere boundaries in the vertebrate hindbrain.

To investigate the potential biological role(s) of the PLZF gene, discovered as a fusion with the RARA locus in a patient with acute promyelocytic leukemia harboring a t(11;17) chromosomal translocation, we have isolated its murine homologue (mPLZF) and studied its patterns of developmental expression. The levels of mPLZF mRNAs increased perinatally in the liver, heart, and kidney, but with the exception of the heart, they were either absent or very low in the adult tissues. In situ analysis of mPLZF expression in mouse embryos between 7.0 and 10.5 days of development revealed that mPLZF mRNAs and proteins were coexpressed in spatially restricted and temporally dynamic patterns in the central nervous system. In the hindbrain region, a segmental pattern of expression correlated with the development of the rhombomeres. From 9.0 days of development, starting first in rhombomeres 3 and 5, there was an ordered down-regulation of expression in the center of each rhombomere, so that 1 day later elevated levels of mPLZF mRNAs and proteins were restricted to cells surrounding the rhombomeric boundaries. The chicken homologue of the PLZF gene, which we have also cloned, demonstrated a similar segmental pattern of expression in the hindbrain. To date, PLZF represents the only example of a transcription factor with elevated expression at rhombomeric boundaries. The high degree of evolutionary conservation between the patterns of PLZF expression during mammalian and avian central nervous system development suggests that it has an important functional role in the regionalization of the vertebrate hindbrain, potentially regulating boundary cell interactions.

Metabolic studies in a mouse model of hepatorenal tyrosinemia: absence of perinatal abnormalities.

Radiation induced chromosomal deletions at the albino locus in the mouse, lethal when homozygous, cause abnormalities of expression of several unlinked liver specific genes. Recently, the gene encoding FAH was shown to be included in the deletions. Since in humans FAH mutations cause tyrosinemia type I, deletion homozygous mice were suspected of having tyrosinemia. Studies of plasma amino acids did not confirm this suspicion. Also, succinylacetone levels were normal in fetal and newborn livers of deletion homozygotes. The present evidence, therefore, does not support the assumption that the earlier described ultrastructural and enzyme abnormalities in deletion homozygotes are secondary effects of tyrosinemia caused by the deletion of FAH.

Selective loss of a DNase I hypersensitive site upstream of the tyrosine aminotransferase gene in mice homozygous for lethal albino deletions.

Several overlapping chromosomal deletions spanning the albino locus in the mouse cause perinatal lethality when homozygous and a block in the transcriptional induction of various unlinked hepatocyte-specific genes. Studies of such lethal albino deletion homozygotes in perinatal stages revealed a deficiency in the transcriptional inducibility of the tyrosine aminotransferase (TAT) gene by glucocorticoids; yet, glucocorticoid receptor and hormone levels were shown to be unaffected. To identify a molecular defect underlying the failure of inducible expression, we examined the chromatin structure of the TAT gene. Whereas in wild-type animals the TAT promoter becomes DNase I hypersensitive at birth, such hypersensitivity fails to develop in lethal albino deletion homozygotes. By contrast, the deletions do not affect the appearance of three DNase I-hypersensitive sites upstream of the TAT promoter in the liver, nor do they affect two hypersensitive sites upstream of the expressed alpha-fetoprotein gene. These findings demonstrate that the abnormality of chromatin structure identified in lethal albino deletion homozygotes occurs on a highly selective basis. Specifically, normal differentiation of the TAT promoter chromatin appears to depend directly or indirectly on the action and product of a gene mapping within the deleted region.

An overview of developmental genetics in mammals.

Publications in mammalian developmental genetics during the past year reflect a shift of emphasis from the phenotypic level to the primary level of gene expression and the nature of the gene product. A result of this, the developmental role of a considerable number of regulatory genes and specific gene sequences have been identified. The cell type-specific effects of several mutations analyzed in the past have been correlated with effects on growth factors and signal transduction pathways. Specific gene sequences such as those containing the homeobox domains and paired-box sequences have recently been implicated in the control of pattern formation and positional information.

Evidence for regulatory genes on mouse chromosome 7 that affect the quantitative expression of proteins in the fetal and newborn liver.

A series of deletions around the albino locus on mouse chromosome 7 is believed to include one or more regulatory genes that control the activities of a cluster of liver enzymes. To further characterize the functions of this region of the mouse genome, we have used quantitative two-dimensional electrophoresis to analyze the effects of two of these deletions, c3H and c14CoS, on the expression of liver proteins. More than 400 distinct protein gene products were quantitated in livers from fetal and newborn wild-type homozygous (cch/cch), heterozygous (cch/c3H or cch/c14CoS), and deletion homozygous (c3H/c3H or c14CoS/c14CoS) mice. Livers of fetal and newborn c3H heterozygotes and homozygous wild-type littermates produced qualitatively identical protein patterns after two-dimensional electrophoresis. In livers of c3H homozygous fetuses, however, abnormal amounts (either increased or decreased relative to homozygous wild-type and heterozygous littermates) of 29 proteins were found. Twenty-eight of these 29 protein anomalies were also found in livers of newborn c3H homozygotes. Livers of fetal and newborn mice homozygous for the c14CoS deletion, which overlaps the c3H deletion and produces a similar phenotype, expressed normal amounts of these proteins. One of the 29 proteins (MSN807) has an amino-terminal sequence similar to a 23-kDa translationally controlled protein abundant in mouse erythroleukemia and sarcoma-180 cells. These results suggest that normal chromosome 7 contains genes, located within the region of the c3H but not the c14CoS deletion, that regulate the abundance of specific proteins in the liver. These proteins cannot be related to the phenotypic alterations shared by the c3H and c14CoS deletions.

Regulatory genes linked to the albino locus in the mouse confer competence for inducible expression on the structural gene encoding serine dehydratase.

A cluster of unlinked genes encoding gluconeogenic enzymes in the mouse is characterized by the failure of normal hormone-inducible expression in animals homozygous for one of several overlapping deletions mapping on chromosome 7 near the albino locus. Previous investigations have shown hormones and their receptors to be normal in the mutants and therefore not responsible for the abnormalities of inducibility. Instead, these studies have implicated a possible failure of the affected structural enzyme genes themselves to attain during prenatal development the competence for inducible gene expression. The results reported here add serine dehydratase (EC 4.2.1.13) and its structural gene to the affected group of gluconeogenic enzymes and their genes. Even though, in deletion homozygotes, serine dehydratase is expressed normally on the constitutive level, hormone-inducible expression fails to develop. The abnormality appears to reside in a defect of prenatal differentiation of cis-acting regulatory elements of the structural gene essential in the pathway of inducible gene expression.

Lethal chromosomal deletions in the mouse, a model system for the study of development and regulation of postnatal gene expression.

Mechanisms involved in the regulation of development and its genetic control are receiving ever-increasing attention in studies of mammalian developmental genetics. The potential success of such studies is strongly enhanced by the availability of suitable systems of analysis. Such a system was identified in a series of radiation-induced chromosomal deletions at and around the albino (c) locus of the mouse associated with cell type-specific effects on liver differentiation. Their detailed study has aided the analysis of possible mechanisms of cell type-specific gene expression. As summarized in this review, the experimental results strongly suggest that specific trans-acting developmental regulatory genes are concerned with the differentiation of hormone-inducible expression of a cluster of hepatocyte specific structural genes mapping in different parts of the genome.

The glucocorticoid hormone signal transduction pathway in mice homozygous for chromosomal deletions causing failure of cell type-specific inducible gene expression.

Wild-type newborn mice are characterized by the ability of certain liver-specific genes encoding various enzymes and mapping on different chromosomes to respond to glucocorticoid induction. Newborn mice homozygous for deletions at and around the albino locus on chromosome 7 fail to develop this competence for hormone-inducible gene expression even through they do show normal constitutive expression of the same genes. Studies of the glucocorticoid hormone signal transduction pathway reported here show identical expression of glucocorticoid receptor mRNA and protein in deletion homozygotes and normal littermates. Furthermore, the receptor interacts normally with the 90-kDa heat shock protein hsp90. Elevated glucocorticoid hormone levels in newborn deletion homozygotes, most likely resulting from their stressed condition, provide an explanation for the reduced binding activities of receptors reported previously. The elimination of receptors and hormones as direct targets of the chromosomal deletion effects suggests that the failure of inducible gene expression might reside in defective competence of the affected structural genes to respond to the hormonal stimulus.

Developmental regulation of constitutive and inducible expression of hepatocyte-specific genes in the mouse.

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of certain liver-specific enzymes in newborn deletion homozygotes. Among these enzymes are L-tyrosine:2-oxoglutarate aminotransferase (EC 2.6.1.5) and phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lase (transphosphorylating); EC 4.1.1.32]. The studies reported here show that in fetal stages constitutive expression of the relevant genes on the level of steady-state mRNA is identical in the livers of homozygous deletion mutants and normal littermates. Furthermore, prenatally these enzymes are expressed also in cell types other than hepatocytes. Thus, the putative trans-acting regulatory factors encoded in the deleted region of chromosome 7 of the mouse appear to be concerned specifically with the regulation of cell type-specific inducible expression of various hepatocyte-specific genes, whereas constitutive expression of the same genes is not affected.

Metallothionein mRNA expression in mice homozygous for chromosomal deletions around the albino locus.

Deletions in chromosome 7 of the mouse affect the expression of the metallothionein gene Mt-1, which maps on chromosome 8, and steady-state levels of Mt-1 mRNA are reduced to 15-40% of normal in livers of newborn mice homozygous for either the c3H or c14CoS deletion. Glucocorticoids fail to induce hepatic Mt-1 mRNA levels in deletion homozygotes in contrast to normal littermates. However, zinc chloride is effective in inducing Mt-1 mRNA levels in livers of deletion homozygotes as well as of their normal littermates. Other tissues (e.g., kidney and intestine) of deletion homozygotes express basal levels of Mt-1 mRNA higher than those of normal littermates. In the intestine these are furthermore inducible by both hormonal and metal agents. Thus, loss of inducibility of the Mt-1 gene in deletion homozygotes concerns glucocorticoids only and is furthermore restricted to specific cell types (i.e., hepatocytes). The trans-acting factor(s) normally encoded in the deleted region of chromosome 7 appears to be instrumental in conferring on the metallothionein gene in hepatocytes the essential competence to respond to hormonal inducing signals.

Trans regulation of the phosphoenolpyruvate carboxykinase (GTP) gene, identified by deletions in chromosome 7 of the mouse.

Livers from newborn mice homozygous for either one of the lethal deletions c14CoS or c3H in chromosome 7 have drastically reduced levels of cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] activity when compared with normal littermates. The structural gene for the enzyme maps on chromosome 2 and appears intact and not grossly rearranged in deletion homozygotes. These mice also have negligible levels of hepatic mRNA encoding this enzyme. Studies of the transcription rate of the gene showed that it was reduced to 25-50% of normal in hepatic nuclei obtained from mice homozygous for either deletion. We suggest that, in addition to the reduction in the level of transcription, the deletions in chromosome 7 may also cause alterations in messenger stability, processing, or transport from the nucleus.

Deletions near the albino locus on chromosome 7 of the mouse affect the level of tyrosine aminotransferase mRNA.

Overlapping chromosomal deletions at the albino locus on chromosome 7 of the mouse affect the expression of several liver enzymes, including tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5). With cloned TAT DNA the integrity of the TAT structural gene and its expression and inducibility by glucocorticoids and cAMP were examined in deletion homozygous mice. No difference in the structure of the gene between normal and mutant mice was detected by Southern blotting. Severely reduced amounts of TAT mRNA were detected in homozygous mutants. The residual mRNA levels could not be modulated by glucocorticoids or cAMP. We conclude that a trans-acting control function required for expression and inducibility of mouse TAT can be assigned to the chromosomal region near the albino locus.

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.

Correction of a genetically caused enzyme defect by somatic cell hybridization.

Liver cells obtained from newborn mice homozygous for any one of several overlapping deletions in chromosome 7 fail to express a number of liver-specific differentiated traits. Among these is the activity of the membrane-bound liver-specific enzyme glucose-6-phosphatase (Glc-6-Pase; D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). Previous studies have led to the suggestion that the region of the genome covered by these deletions includes genes that normally regulate the expression of structural genes encoding liver-specific enzymes and proteins mapping elsewhere in the genome. To find out whether the deficiency of Glc-6-Pase may be caused by the deletion of the relevant structural gene, mouse liver cells homozygous for the deletion c14CoS were hybridized with 2S Faza rat hepatoma cells, and the hybrid cell cultures were analyzed for mouse and rat Glc-6-Pase activity. Hybrids showed expression of mouse Glc-6-Pase activity, proving that the structural gene for this enzyme is not included in the deletion c14CoS in chromosome 7. In the hybrid cells the rat hepatoma genome apparently contributes a factor that activates the structural gene of the mouse and corrects its failure of expression, which most likely resulted from the deletion of an essential regulatory or processing gene. By using as a marker glucose-6-phosphate isomerase (Glc-6-PIase; glucosephosphate isomerase, D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9), known to map on chromosome 7, this entire chromosome could be excluded as a possible carrier of the Glc-6-Pase structural gene. In addition, the structural genes for Glc-6-Pase and for tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), another enzyme deficient in lethal deletion homozygotes, were shown to map on two different chromosomes. Together with our previous studies of TyrATase gene regulation, the present experiments suggest that the region of the mouse genome defined by the deletions includes one or more genes regulating the expression of several structural genes that map on different chromosomes and that encode liver-cell-type specific traits.

Epidermal growth factor and glucagon receptors in mice homozygous for a lethal chromosomal deletion.

The binding of epidermal growth factor (EGF) and of glucagon to their receptors has been examined in single-cell suspensions obtained from livers and other organs of newborn mice homozygous for a perinatally lethal deletion that includes the albino (c) locus on chromosome 7. Competition experiments with 125I-labeled and nonradioactive EGF and Scatchard analysis of equilibrium binding data showed that hepatocytes from deletion homozygotes had only approximately equal to 20% of the number of specific EGF receptors present in cells from normal littermates. In contrast, EGF binding to single-cell suspensions from organs other than the liver was normal in deletion homozygotes. Similar results were obtained in competitive displacement experiments with 125I-labeled and nonradioactive glucagon: hepatocytes from deletion mutants showed only approximately equal to 30% of the specific glucagon binding sites found in cells from normal littermates. As in the case of EGF, the decreased binding was due to decreased numbers of glucagon receptors per cell rather than alterations in receptor affinity, and glucagon binding to single-cell suspensions from organs other than the liver was normal in the deletion mutants. The reductions in numbers of EGF and glucagon receptors are liver-cell specific as are the previously described ultrastructural and biochemical abnormalities in these mutants. The significance of cell membrane integrity and hormone-receptor interactions in the control of normal liver cell differentiation is discussed.

Recessive lethal deletion on mouse chromosome 7 affects glucocorticoid receptor binding activities.

The hormone binding activity of glucocorticoid receptors is decreased by approximately equal to 75% in the livers of mice homozygous for c14CoS, one of several overlapping radiation-induced deletions on chromosome 7. These deletions have been shown previously to map at and around the albino (c) locus and to act as recessive lethals. They are associated with intractable hypoglycemia, ultrastructural abnormalities in certain liver cell membranes, and deficiencies of specific liver proteins and enzymes that are induced by insulin or glucocorticoids, or both. Scatchard analysis of [3H]dexamethasone binding to receptors in control and mutant liver extracts revealed that the glucocorticoid binding constants were similar; however, the control extracts bound approximately equal to 4 times as much steroid as did mutant extracts. Analysis by DNA-cellulose column chromatography demonstrated that the ability of activated glucocorticoid receptors to form complexes with DNA appears to be unaltered in the mutants; both mutant and control cytosols contain glucocorticoid receptors that elute from DNA-cellulose columns at two different salt concentrations. Sucrose density gradient centrifugation revealed the glucocorticoid receptors in mutant livers to have approximately the same sedimentation coefficient as receptors in control livers. The decrease in glucocorticoid binding activity in mutant liver cell extracts is comparable in degree to that previously reported for insulin binding activity. The gene sequences deleted in the homozygous deletion mutants appear to be instrumental in the regulation rather than the structural determination of both glucocorticoid and insulin receptors.